Recent studies performed in our laboratory have identified novel roles for the pleckstrin homology domain interacting protein (PHIP) gene as a marker and mediator of melanoma progression. Increased PHIP levels were an independent predictor of melanoma survival. PHIP was activated in triple-negative melanomas (harboring wild type BRAF, NRAS and PTEN), thereby helping to characterize this poorly understood molecular subset of melanoma. In addition, shRNA-mediated down regulation of PHIP significantly suppressed the invasiveness and metastatic potential of both murine and human melanoma, including in triple-negative melanoma cell lines. In this proposal, we plan to confirm the important role played by PHIP as a biomarker for melanoma and its role as a mediator of melanoma progression. Our specific aims are: (1) to validate the prognostic role of PHIP in melanoma. We will assess the prognostic role of PHIP in a distinct cohort of 250 patients with primary melanoma amassed from a different population. We will determine PHIP levels using both immunohistochemical analysis and fluorescence in situ hybridization and correlate PHIP levels with survival. We will also assess PHIP levels in 200 melanoma metastases. This aim will validate the prognostic impact of PHIP in melanoma, and determine its role as a melanoma progression marker. (2) To assess the role of PHIP in the molecular classification of melanoma. We will examine the 450 melanomas with documented PHIP levels for the most commonly acquired mutations in melanoma, including BRAF, NRAS, and PTEN. This aim will serve to identify the molecular subtypes of melanoma with activated PHIP levels. It will also determine the proportion of triple-negative melanomas that have activated PHIP levels. (3) To validate the functional role of PHIP in the progression of human melanoma. We will examine the role of PHIP in mediating human melanoma progression. We will assess the consequences of both shRNA-mediated PHIP knockdown as well as PHIP overexpression in the progression of early-passage human melanoma cultures with defined BRAF mutational status. If successful, these studies will validate the role of PHIP as a novel molecular prognostic factor for melanoma and establish its utility in the molecular classification of this disease. Finally, they will validate he functional role of PHIP in melanoma progression, establishing it as a viable target for therapy.